Genetic evaluation of bone marrow-derived mesenchymal stem cells by a modified karyotyping method

Abstract

Bone marrow is the most important source of mesenchymal stem cells (MSCs). Significant development in the comprehension of MSCs biology has opened the way to their clinical use. The object of this study was to establish a method for karyotyping of canine MSCs. We examined the effect of the increase in passage number on the chromosome and appearance of MSCs. A large 3-year-old mixed breed dog was used. Bone marrow (20 ml) was collected from the proximal humerus under general anesthesia and cultured and passaged in alpha minimal essential medium (αMEM) and fetal bovine serum (FBS). The canine bone marrow MSCs (BM-MSCs) were characterized by reverse transcription polymerase chain reaction (RT-PCR) and flask adherent fibroblast-like morphology. Karyotyping of cells of passages 1 and 8 was performed. The chromosome numbers were counted and evaluated to obtain the percentage of diploid cells. The best time for exposing BM-MSCs with media containing colcemid was 10 h. These cells were positive for the BM-MSCs markers CD90 and negative for hematopoietic markers CD34 and CD45 by RT-PCR. Karyotyping of these cells was normal, and also fibroblast-like cells were observed in every passage.